Structural Characterization of Intramolecular Hg 2+ Transfer between Flexibly Linked Domains of Mercuric Ion Reductase
Structural Characterization of Intramolecular Hg 2+ Transfer between Flexibly Linked Domains of Mercuric Ion Reductase
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DOI：
10.1016/j.jmb.2011.08.042
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发表时间：
2011-10-28
影响因子：
5.6
通讯作者：
Miller, Susan M.
中科院分区：
文献类型：
--
作者：
Johs, Alexander;Harwood, Ian M.;Miller, Susan M.
汞离子还原酶MerA是由mer操纵子编码的细菌耐汞性的核心组分。许多MerA蛋白具有金属伴侣样N-末端结构域（NmerA），其可以将Hg 2+转移到催化核心结构域（Core）以还原为Hg-0。这些结构域通过类似于易受蛋白水解影响的30个残基的接头连接到同源二聚体核心，后者阻止了全长蛋白中NmerA和核心相互作用的表征。在这里，我们报告纯化的同质全长MerA从Tn 21 mer操纵子使用融合蛋白的构建和联合收割机结合小角X射线散射和小角中子散射与分子动力学模拟来表征全长野生型和突变体MerA蛋白的结构，模仿系统之前和期间的Hg 2+从NmerA的核心。回转半径，距离分布函数，和Kratky图来自小角X-射线散射数据是一致的全长MerA采用细长的构象作为一个结果的灵活性，在连接器的NmerA域。最好的散射配置文件复制使用一个合奏的连接器构象。NmerA的这种柔性附着可以促进从不同蛋白质底物中快速有效地去除Hg 2+。使用一个特定的突变体的MerA允许形成的金属介导的相互作用NmerA和核心和确定的位置和相对取向的NmerA的核心在Hg 2+切换。(C)2011爱思唯尔有限公司保留所有权利。
The enzyme mercuric ion reductase MerA is the central component of bacterial mercury resistance encoded by the mer operon. Many MerA proteins possess metallochaperone-like N-terminal domains (NmerA) that can transfer Hg2+ to the catalytic core domain (Core) for reduction to Hg-0. These domains are tethered to the homodimeric Core by similar to 30-residue linkers that are susceptible to proteolysis, the latter of which has prevented characterization of the interactions of NmerA and the Core in the full-length protein. Here, we report purification of homogeneous full-length MerA from the Tn21 mer operon using a fusion protein construct and combine small-angle X-ray scattering and small-angle neutron scattering with molecular dynamics simulation to characterize the structures of full-length wild-type and mutant MerA proteins that mimic the system before and during handoff of Hg2+ from NmerA to the Core. The radii of gyration, distance distribution functions, and Kratky plots derived from the small-angle X-ray scattering data are consistent with full-length MerA adopting elongated conformations as a result of flexibility in the linkers to the NmerA domains. The scattering profiles are best reproduced using an ensemble of linker conformations. This flexible attachment of NmerA may facilitate fast and efficient removal of Hg2+ from diverse protein substrates. Using a specific mutant of MerA allowed the formation of a metal-mediated interaction between NmerA and the Core and the determination of the position and relative orientation of NmerA to the Core during Hg2+ handoff. (C) 2011 Elsevier Ltd. All rights reserved.